Application of phosphatidyl ethanolamine as respiratory syncytial virus vaccine metabolic adjuvant

Abstract

The invention relates to the technical field of vaccines and adjuvants, in particular to application of phosphatidyl ethanolamine as a respiratory syncytial virus vaccine metabolic adjuvant. The phosphatidyl ethanolamine, as a metabolic adjuvant of the RSV vaccine, can regulate T cell immune response and prevent VED, so that balanced immune memory is generated in the body of a vaccinator, and when RSV infection occurs, protection is provided while vaccine enhanced inflammatory diseases are prevented, namely, the phosphatidyl ethanolamine is safe and effective.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines and adjuvants, and in particular, to the use of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccines. Background Art

[0002] Respiratory syncytial virus (RSV) can cause lower respiratory tract infections in infants, the elderly, and immunocompromised patients, and is one of the important pathogens of respiratory tract infections. Vaccines are the most effective means to solve pathogen infections. However, in the clinical study of the formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine developed in the 1960s, when infants and young children who had been vaccinated with this vaccine were naturally infected with RSV, more severe pneumonia occurred, and two deaths were caused, namely vaccine-enhanced disease (VED). A large number of inflammatory cell infiltrations and Th2-type immune responses were presented in the lungs of the deceased. This incident has delayed the development of RSV vaccines for decades, and the safety issue represented by VED has become the primary problem to be solved in the development of RSV vaccines.

[0003] In traditional concepts, only adaptive immunity has the characteristic of immune memory. Recently, it has been found that innate immune cells, such as macrophages (MΦ), natural killer cells (NK), dendritic cells (DC), etc., can produce enhanced or tolerant responses when stimulated the same or differently, which is called innate immune memory. Its mechanism is different from the recombination and clonal expansion of immunoglobulin family genes in adaptive immunity, but depends on epigenetic modification, transcriptional changes, and metabolic reprogramming after stimulation to produce memory. However, its role has been emphasized in the processes of inflammatory diseases, tumors, and vaccine development.

[0004] Adjuvants are important components of vaccines, which can enhance the immune response of the body to vaccines or change the type of immune response. By rationally designing adjuvants, the protective effect of vaccines can be enhanced, and the toxic and side effects of vaccines can be inhibited by regulating the type of immune response, thereby improving its safety. Developing an ideal RSV vaccine adjuvant to inhibit the occurrence of VED by regulating the immune balance is an important measure to solve the safety problem of RSV vaccines; traditional adjuvants still mainly rely on a trial-and-error method and lack targeting. The development of new adjuvants uses systems vaccinology to conduct mechanism research on inactivated RSV vaccines and design targeted adjuvants, such as metabolic adjuvants, signaling pathway adjuvants, etc.

[0005] Based on the above background, the present application proposes that phosphatidylethanolamine can be used as a combined metabolic adjuvant for FI-RSV + aluminum salt adjuvant, with the expectation of developing a safe and reliable RSV vaccine.

[0006] In the prior art, there have been no relevant reports and studies on using phosphatidylethanolamine as a metabolic adjuvant for RSV vaccines. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccines.

[0008] To solve the above problems, the technical solutions adopted by the present invention are as follows: Technical Theme 1 Application of phosphatidylethanolamine as a metabolic adjuvant for RSV vaccines.

[0009] As a further improvement of the present invention, the RSV vaccine contains or does not contain an aluminum salt adjuvant.

[0010] As a further improvement of the present invention, the RSV vaccine is an inactivated RSV vaccine or a subunit RSV vaccine that induces a Th2-type response. As a further improvement of the present invention, the inactivated RSV vaccine is a formalin-inactivated RSV vaccine or a β-propiolactone-inactivated RSV vaccine.

[0011] As a further improvement of the present invention, the application is that phosphatidylethanolamine is injected into the body before or simultaneously with the vaccine to induce balanced immune memory.

[0012] As a further improvement of the present invention, the application is that the phosphatidylethanolamine adjuvant is administered the same or different times and by the same or different routes as the vaccine.

[0013] The beneficial effects of adopting the above technical solutions are as follows: The present invention provides an application of phosphatidylethanolamine as a metabolic adjuvant for respiratory syncytial virus vaccines. As a metabolic adjuvant for RSV vaccines, phosphatidylethanolamine regulates T cell immune responses, prevents the occurrence of VED, enables the generation of balanced immune memory in vaccine recipients, and when the corresponding pathogen infects, it provides protection while preventing the occurrence of vaccine-enhanced inflammatory diseases, that is, it achieves both safety and effectiveness. Brief Description of the Drawings

[0014] Figure 1This is a diagram showing the changes in H&E staining and PAS staining of lung tissues in each group of BALB / c mice treated with the PBS group, FI-RSV group, FI-RSV + low (L, 2 μg / mouse), medium (M, 10 μg / mouse), and high (H, 50 μg / mouse) dose PE groups in Example 1 of the present invention after 4 days of intranasal instillation. In the figure, the upper row is H&E × 200, and the lower row is PAS × 200. FI-RSV is a formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); PE is phosphatidylethanolamine; L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine; Figure 2 This is a statistical chart showing the changes in H&E staining and PAS staining of lung tissues in each group of BALB / c mice treated with the PBS group, FI-RSV group, FI-RSV + low (L), medium (M), and high (H) dose PE groups in Example 1 of the present invention after 4 days of intranasal instillation. The left figure is the inflammation score, and the right figure is the mucus score. In the figure, FI-RSV is a formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05, **: P <0.01, ***: P <0.001); Figure 3 This is a diagram showing the white blood cell count in bronchoalveolar lavage fluid in each group of BALB / c mice treated with the PBS group, FI-RSV group, FI-RSV + low (L), medium (M), and high (H) dose PE groups in Example 1 of the present invention after 4 days of intranasal instillation. In the figure, FI-RSV is a formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05, **: P <0.01, ***: P <0.001); Figure 4 This is a diagram showing the expression of RSV-N in lung tissues of each group detected by real-time quantitative PCR after 4 days of RSV challenge in BALB / c mice treated with the PBS + RSV group, FI-RSV group, FI-RSV + low (L), medium (M), and high (H) dose PE groups in Example 1 of the present invention. In the figure, FI-RSV is a formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05, **: P <0.01, ***: P<0.001); Figure 5 In Example 1 of the present invention, BALB / c mice were treated with the PBS group, the FI-RSV group, and the FI-RSV + low (L), medium (M), and high (H) dose PE groups, and after 4 days of intranasal administration, the expression of cytokine mRNA in the lung tissues of each group was detected by real-time quantitative PCR. In the figure, FI-RSV is a formalin-inactivated RSV vaccine (containing aluminum salt adjuvant); L-PE is low-dose phosphatidylethanolamine; M-PE is medium-dose phosphatidylethanolamine; H-PE is high-dose phosphatidylethanolamine (*: P <0.05, **: P <0.01, ***: P <0.001). Detailed implementation manners

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0016] The FI-RSV vaccine used in this application was prepared through the steps described in Preparation Example 1.

[0017] Preparation Example 1 (1) Inactivation: Take the virus solution prepared by infecting vero cells with RSV, add 5 μl of formalin (final dilution 1:4000) to the centrifuge tube containing the RSV virus solution, blow and suck several times, and incubate in a 37°C shaker for 72 hours. (Add 25 μl of 40% formaldehyde solution to every 100 ml of virus cell solution) (2) Impurity removal: After 72 hours, take out the centrifuge tube, centrifuge at 4100 rpm (1800 g) at 4°C for 10 min; take the supernatant for step (3).

[0018] (3) Ultracentrifugation: Take the supernatant, centrifuge at 49943 g at 4°C for 60 min, retain the precipitate, and discard the supernatant.

[0019] (4) Ultracentrifugation: Resuspend the precipitate in step (3) with PBS, perform ultracentrifugation, centrifuge at 49500 g at 4°C for 60 min, retain the precipitate, and discard the supernatant.

[0020] (5) Vaccine collection: Resuspend the precipitate in (4) with PBS (1 / 25 of the original volume), mix it with an aluminum hydroxide solution with a final concentration of 4 mg / ml. In this application, the volume of PBS used to resuspend the precipitate is 4 ml, the concentration of the aluminum hydroxide solution is 10 mg / ml, and the volume is 2.67 ml. Vortex and mix well, and shake at 250 rpm overnight at room temperature. Then centrifuge at 2800 rpm, discard the supernatant, and resuspend the precipitate in 1 / 4 volume of PBS. Aliquot into EP tubes (1 ml / tube) and store at -80°C for later use.

[0021] Study on Phosphatidylethanolamine as a Metabolic Adjuvant for RSV Inactivated Vaccine Experimental study on immunizing BALB / c mice with formalin-inactivated RSV vaccine (FI-RSV, containing aluminum salt adjuvant) + phosphatidylethanolamine (subsequently referred to as PE) adjuvant at low (L, 2 μg / mouse / time), medium (M, 10 μg / mouse / time), and high (H, 50 μg / mouse / time) doses 1.1 Grouping of BABL / c mice (6 mice / group): ① PBS group; ② PBS + RSV group; ③ FI-RSV group; ④ FI-RSV + L-PE group; ⑤ FI-RSV + M-PE group; FI-RSV + H-PE group; 1.2 Vaccine inoculation and RSV challenge protocol for BABL / c mice: Collect 6 - 8-week-old BALB / c mice, and intramuscularly inoculate each group with the respective vaccines and adjuvants on days 0, 14, and 28. During injection, first intramuscularly inject the PE adjuvant, and after a 15-minute interval, inject the formalin-inactivated RSV vaccine (FI-RSV, containing aluminum salt adjuvant). The injection volume of the formalin-inactivated RSV vaccine is 50 μl / mouse / time. For the groups that do not need to inject the vaccine or adjuvant, inject an equal volume of PBS. On day 42, the PBS group is instilled with PBS through the nose, and the other groups are instilled with RSV through the nose. On day 47, sacrifice the mice in each group for sampling. Perform white blood cell counting on the bronchoalveolar lavage fluid, take lung tissues, embed them in paraffin, section them, and stain with H&E and PAS; take some lung tissues, extract RNA, and perform real-time quantitative PCR detection.

[0022] The results showed 1. The pulmonary tissue inflammation pathology in the adjuvant groups was significantly inhibited (such as Figure 1 and Figure 2 ) Visible from the H&E staining of the pulmonary tissue pathological sections: In the FI-RSV + L-PE group, there was a small amount of inflammatory cell infiltration in the local bronchi, and a small part of the alveoli fused. The inflammation score was significantly lower than that of the FI-RSV group (<0.01), and significantly higher than that of the PBS group (<0.01); in the FI-RSV + M-PE group, there was an extremely small amount of inflammatory cell infiltration in the local bronchi, and occasional alveolar fusion. The inflammation score was significantly lower than that of the FI-RSV group (<0.001), and there was no significant difference from the PBS group ( P <0.01), significantly higher than that of the PBS group ( P <0.01); in the FI-RSV + M-PE group, there was an extremely small amount of inflammatory cell infiltration in the local bronchi, and occasional alveolar fusion. The inflammation score was significantly lower than that of the FI-RSV group (<0.001), and there was no significant difference from the PBS group ( P <0.001), and there was no significant difference from the PBS group ( P> 0.05); There was a very small amount of inflammatory cell infiltration in the local bronchi of the FI-RSV+H-PE group, and occasional alveolar fusion was seen. The inflammation score was significantly lower than that of the FI-RSV group ( P <0.001), and there was no significant difference from the PBS group ( P <0.01); There was no statistical difference in the inflammation scores among the three groups of FI-RSV+L-PE group, FI-RSV+M-PE group and FI-RSV+H-PE group ( P > 0.05).

[0023] PAS staining of lung tissue pathological sections showed that: There was no obvious mucus secretion around the bronchi in the FI-RSV+L-PE group. The mucus secretion score was significantly lower than that of the FI-RSV group ( P <0.01), and there was no significant difference from the PBS group ( P > 0.05); There was no obvious mucus secretion around the bronchi in the FI-RSV+M-PE group. The mucus secretion score was significantly lower than that of the FI-RSV group ( P <0.001), and there was no significant difference from the PBS group and the FI-RSV+L-PE group ( P > 0.05); There was no obvious mucus secretion around the bronchi in the FI-RSV+PE group. The mucus secretion score was significantly lower than that of the FI-RSV group ( P <0.001), and there was no statistical difference from the PBS group, the FI-RSV+L-PE group and the FI-RSV+M-PE ( P > 0.05).

[0024] It was proved that when reinfected with RSV after vaccination, phosphatidylethanolamine as a metabolic adjuvant could significantly inhibit the inflammation and mucus secretion of lung tissue.

[0025] 2. The number of inflammatory cells in the bronchoalveolar lavage fluid of the adjuvant group was significantly reduced (such as Figure 3 ). The BALF white blood cell count in the FI-RSV+L-P group was significantly lower than that in the FI-RSV group ( P <0.001), and significantly higher than that in the PBS group ( P <0.01). The BALF white blood cell count in the FI-RSV+M-PE group was significantly lower than that in the FI-RSV group ( P <0.001), and significantly higher than that in the PBS group ( P <0.01). The BALF white blood cell count in the FI-RSV+H-PE group was significantly lower than that in the FI-RSV group ( P <0.001), and significantly higher than that in the PBS group ( P <0.01). There was no statistical difference among the FI-RSV+L-PE group, the FI-RSV+M-PE group and the FI-RSV+H-PE group ( P> 0.05).

[0026] It was demonstrated that phosphatidylethanolamine, as a metabolic adjuvant, could significantly inhibit BALF leukocytosis when RSV reinfection occurred after vaccination.

[0027] 3. Expression of RSV-N in mouse lung tissue with PE as a metabolic adjuvant (as Figure 4 ) In the groups applying low, medium, and high doses of PE, the RSV viral load in lung tissue was significantly lower than that in the PBS group and FI-RSV. The expression of the RSV housekeeping gene RSV-N in the lung tissue of mice in the FI-RSV+H-PE group was significantly lower than that in the PBS group ( P <0.001) and the FI-RSV group ( P <0.01).

[0028] It was demonstrated that phosphatidylethanolamine, as a metabolic adjuvant, could also significantly inhibit the proliferation of RSV in the lungs when RSV reinfection occurred after vaccination.

[0029] 4. Balance of Th1 and Th2 type responses in the lung tissue of the medium and high dose adjuvant groups ( Figure 5 ) For the lung tissue cytokine mRNA in the groups applying low, medium, and high doses of PE, the mRNA of the Th1 type cytokine IFN-γ was significantly lower than that in the FI-RSV group and the PBS group, and the mRNA expression of the Th2 type cytokine IL-4 was significantly lower than that in the FI-RSV group ( P <0.01), with no significant difference from the PBS group ( P > 0.05). The mRNA expression of IL-13, a Th17 type cytokine, was significantly lower than that in the FI-RSV group (P < 0.01), with no significant difference from the PBS group ( P > 0.05); the mRNA expression of the Th17 type cytokine IL-17 in the FI-RSV+H-PE group was significantly lower than that in the FI-RSV group ( P <0.001), with no statistical difference from the PBS group.

[0030] It was demonstrated that phosphatidylethanolamine, as a metabolic adjuvant, could balance and regulate the T cell immune response, prevent the occurrence of VED, and generate balanced immune memory in vaccinated mice when RSV reinfection occurred after vaccination.

[0031] All of the above experimental results showed that PE, as a metabolic adjuvant for FI-RSV, could improve its protective effect and safety.

[0032] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of phosphatidylethanolamine as a metabolic adjuvant for RSV vaccines.

2. The application according to claim 1, characterized in that The RSV vaccine contains or does not contain an aluminum salt adjuvant.

3. The application according to claim 1, characterized in that, The RSV vaccine is an inactivated RSV vaccine or a subunit RSV vaccine that induces a Th2-type response.

4. The application according to claim 3, characterized in that The inactivated RSV vaccine is a formalin-inactivated RSV vaccine or a β-propiolactone-inactivated RSV vaccine.

5. The application according to claim 1, characterized in that, The use is that phosphatidylethanolamine is injected into the body either in advance or simultaneously with the vaccine to induce balanced immune memory.

6. The application according to claim 1, characterized in that, The use is that the phosphatidylethanolamine metabolic adjuvant is administered the same or different number of times and by the same or different routes as the vaccine.

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